WO1994019371A1 - Nouveau peptide presentant une activite inhibitrice de l'elastase et son procede de production - Google Patents

Nouveau peptide presentant une activite inhibitrice de l'elastase et son procede de production Download PDF

Info

Publication number
WO1994019371A1
WO1994019371A1 PCT/JP1994/000284 JP9400284W WO9419371A1 WO 1994019371 A1 WO1994019371 A1 WO 1994019371A1 JP 9400284 W JP9400284 W JP 9400284W WO 9419371 A1 WO9419371 A1 WO 9419371A1
Authority
WO
WIPO (PCT)
Prior art keywords
gly
glu
cys
leu
phe
Prior art date
Application number
PCT/JP1994/000284
Other languages
English (en)
Japanese (ja)
Inventor
Shigeru Nakano
Toshiyuki Mabuchi
Miki Tada
Yasuo Taoda
Dan Sugino
Yoshio Kono
Kaoru Nishimura
Minoru Okushima
Original Assignee
Nissin Shokuhin Kabushiki Kaisha
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nissin Shokuhin Kabushiki Kaisha filed Critical Nissin Shokuhin Kabushiki Kaisha
Priority to AU61151/94A priority Critical patent/AU676019B2/en
Priority to US08/325,243 priority patent/US5541288A/en
Priority to KR1019940703771A priority patent/KR100213662B1/ko
Priority to EP94907668A priority patent/EP0643075A4/fr
Priority to CA002134240A priority patent/CA2134240C/fr
Publication of WO1994019371A1 publication Critical patent/WO1994019371A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel peptide having a pharmacologically useful activity prepared by modifying a part of amino acids constituting a peptide having a known protease inhibitory activity, and a method for producing the same.
  • Erasase a type of proteinase, is said to play an important role in the metabolism of living tissues in vivo.
  • elastase secreted from neutrophils may increase its secretion in infectious diseases and inflamed tissues, and may be closely involved in defense against infection and degradation and regeneration of injured tissues.
  • proteins that inhibit the activity of elastase are also produced in the living body, and there is also a mechanism that neutralizes excess elastase activity and regulates it so that excessive living tissue is not degraded.
  • the activity of elastase and the activity of elastase inhibitory protein are strictly regulated and homeostasis is maintained.
  • the morbidity is caused.
  • diseases such as emphysema, idiopathic pulmonary fibrosis, and adult respiratory distress syndrome (ARDS) develop in the lungs [metabolism, ⁇ , pp. 41-49 (1992)].
  • ARDS adult respiratory distress syndrome
  • neutrophil elastase activity is closely related to the development of diseases such as rheumatoid arthritis and osteoarthritis [Agents Actions.
  • al-AT ⁇ ⁇ -antiribose
  • inflammation is caused in the lung area by the inflammation-causing substance contained in the contaminated air, and a1-AT is also oxidized by active oxygen released from leukocytes etc. accumulated in the inflamed area, inhibiting elastase. Activity is lost [J. Clin. Invest., 66, pp. 987-995 (1980)]. It is thought that such inactivation of ⁇ 1-AT causes an excess state of elastase in the local lung, and excess elastase degrades alveolar tissue, resulting in pulmonary diseases such as emphysema (chemotherapy). Pp. 1455-1459, (1989)].
  • neutrophils or other leukocytes accumulate at that location to secrete elastase and generate a large amount of active oxygen. Inactivates 1-AT, which results in the collapse of lung tissue [metabolism, pp. 41-49 (1992)].
  • elastase inhibitors such as ⁇ 1-AT
  • Administration of elastase inhibitors, such as ⁇ 1-AT, locally to the lesion site or into the blood is said to be effective in preventing and treating the above-mentioned diseases caused by elastase.
  • elastase inhibitors such as ⁇ 1-AT
  • spray-inhalation method using a nebulizer is widely used as a method of administering a therapeutic agent for pulmonary disease, but as described above, ⁇ 1-AT is extremely susceptible to oxidation and inhibits elastase during spraying. There is a phenomenon that the activity is inactivated by air oxidation, and even when administered by other administration methods, it has the disadvantage that it is oxidized and inactivated by active oxygen generated from various cells in vivo. Was.
  • an elastase inhibitor is considered to be extremely useful for the prevention and treatment of elastase-induced diseases such as emphysema, but elastase inhibitory proteins derived from humans such as 1-AT.
  • Low quality and chemical synthesis Peptide elastase inhibitors have the above-mentioned various problems and have not been clinically applied as pharmaceuticals until now.
  • the present invention has been made in view of the above-mentioned problems in the technical field, and has as its object the purpose of which has already been marketed as a pharmaceutical and its safety against humans has been confirmed.
  • Amino acid of the C-terminal domain hereinafter, referred to as "HI-8”
  • Ulinastatin urinary tribsin inhibitor
  • An object of the present invention is to provide a novel peptide which is extremely useful pharmacologically as a therapeutic or prophylactic agent for the present invention, and a method for producing the same by genetic engineering using recombinant microorganisms.
  • human urinary trypsin inhibitor which is produced by proteolysis in vivo or limited degradation of UTI in excess of trypsin in vitro, and has a trypsin inhibitory activity.
  • human urinary trypsin inhibitor which is produced by proteolysis in vivo or limited degradation of UTI in excess of trypsin in vitro, and has a trypsin inhibitory activity.
  • HI-8 Hoppe-Sey 1 ei ”sZ. Physiol. Chem. Bd. 362 ⁇ S. pp.
  • Novel peptide having a pharmacologically useful elastase inhibitory activity obtained by modifying a part by genetic engineering or protein engineering techniques, and a gene encoding the peptide A recombinant microorganism carrying the gene; and a method for producing the novel peptide using the recombinant microorganism.
  • UTI is a trypsin inhibitor contained in human urine, and a sample purified from urine is used for the treatment of acute circulatory insufficiency and the treatment of acute inflammation, and is used for medical purposes. It is a protein that has already been confirmed to be safe for intermediate administration [Digest. Dis. Sci., 29, pp. 26-32 (1984): Jpn. J.
  • the three-dimensional structure when HI-8 binds to human neutrophil elastase was estimated using a computer program for estimating the three-dimensional structure of the protein, and the binding sites of both were estimated. Based on the consideration of space coordinates, m-8 is not oxidized and A modified site that specifically binds to neutrophil elastase and the type of modified amino acid at that site were assumed.
  • DNA encoding HI-8 and the amino acid sequence of the appropriate secretion signal peptide was chemically synthesized using the codons and cloned into the appropriate expression plasmid. . Then, the plasmid containing the synthetic DNA was subjected to a site-directed mutagenesis treatment so as to match the amino acid sequence assumed from the above-described computer analysis.
  • the K i value for human neutrophils gills static Ichize is, the ones that line Cormorants prior to recombination HI -8 Modifications 6. is approximately 9 X 1 0- 7 M, this peptide 3rd, 11th, 15th, 18th, 46th from N-terminal of -Th ⁇ Mi Bruno acid residues, Ri by the and by Unaa Mi Amino Acids, respectively in the residues converted child shown in the examples, reducing the K i value to a 6 X 10- 1 ° M I was able to do it.
  • the present inventors have found that a strong elastase inhibitory activity can be obtained by intentional modification of a part of the amino acid sequence constituting -8.
  • the general formula of the amino acid sequence of the modified peptide of HI-8 exhibiting the elastase inhibitory activity revealed in the present invention is shown in SEQ ID NO: 1.
  • the amino acid at position 15 in the sequence shown in SEQ ID NO: 1 is Hydrophobic amino acid residues such as amino acids, leucine and valin are preferred, and the amino acid at position 11 is glutamic acid, and the amino acid at position 46 is preferred. It was also evident that the modified peptide with nodal acid as the daryumin inhibited the human neutrophil elastase most potently.
  • This modified HI-8 peptide has an amino acid sequence composed of 66 amino acids shown in SEQ ID NO: 1, but has an N-terminal or C-terminal amino acid sequence. Further deletion of part of the sequence would still exhibit inhibitory activity on human neutrophil elastase. That is, the scope of the present invention naturally encompasses a core structure exhibiting a human neutrophil elastase inhibitory action in a modified amino acid residue of HI-8.
  • amino acid residues are located in the vicinity of the active center of the modified amino acid residue of the present invention, for example, in the N-terminal direction and the C-terminal direction around the amino acid at the 15th position. It is considered that the object of the present invention can also be sufficiently achieved by substituting a peptide composed of amino acid with a corresponding portion in another serine protease inhibitor.
  • the modified HI-8 peptide of the present invention is obtained by chemically synthesizing a DNA encoding the sequence and inserting it downstream of an appropriate promoter, and
  • the transformant can be produced by introducing the transformant into an appropriate host such as Bacillus subtilis or animal cells and culturing the transformant.
  • DNA encoding UTI is isolated from a cDNA library of a suitable human, and the desired modified peptide is coded by introducing necessary mutations into this DNA by site-directed mutagenesis. It is also possible to obtain DNA to be used.
  • Secretion of the modified peptide of the present invention into a culture solution by a recombinant host is useful for efficiently purifying the modified peptide.
  • an appropriate signal sequence for secretion is inserted upstream of the DNA encoding the modified peptide.
  • animal cells when used as a host, they can be secreted into a culture solution, and when the host is Escherichia coli, they can be secreted into a periplasm or a culture solution.
  • the modified peptide of the present invention is also effective to connect the modified peptide of the present invention to another appropriate protein and to produce the host in the form of a stable fusion protein.
  • the fusion protein is purified by utilizing the antigenic properties of the protein portion, etc., and the obtained fusion protein is ligated to the site using a chemical cleavage method appropriate for the sequence of the appropriate protease. By specific cleavage, the desired modified peptide can be released for separation and purification.
  • the modified peptide of the present invention has a suitable promoter and SD sequence such as lac, trc, and tac upstream of a DNA sequence encoding the modified peptide, and a suitable downstream thereof. It can be produced by adding a terminating signal, connecting to a replicable plasmid corresponding to the host, introducing it into a microbial host, and culturing the resulting transformant. When expressing the desired peptide directly in the bacterial cell, ATG, a translation initiation signal, is added to the 5 'end of the gene. When secreting the modified peptide of the present invention into a culture solution or periplasm, a signal is upstream of the gene. Adjacent DNA encoding peptides.
  • the modified peptide of the present invention When the modified peptide of the present invention is produced using a microbial host, an appropriate S--S bond is not formed between cysteine residues, and the modified peptide is expressed without taking an active conformation. There is also. When expressed in the form of an inclusion body as described above, it is advantageous for crude purification, but it is difficult to desire an active modified peptide for the same reason. In such a case, the activity can also be recovered by first purifying the S—S bond in a reduced state with an appropriate reducing agent and then using a reoxidation method suitable for the characteristics of the peptide.
  • the modified peptide obtained by the present invention can be easily separated and purified from a reaction solution, a bacterial cell or a culture solution by combining known separation and purification methods.
  • separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • differences in molecular weight such as ion exchange chromatography, methods using charged affinity, methods using hydrophobic differences, such as reversed phase chromatography, or isoelectric focusing
  • a method utilizing the difference between isoelectric points, such as an electrophoresis method can be adopted.
  • FIG. 1 is a diagram showing 10 oligonucleotide cleavage sites in a gene designed to have the structure shown in SEQ ID NO: 2 c
  • FIG. 2 is a diagram showing a process of constructing the expression plasmid of the present invention.
  • Example 1 Code the peptide (H8) on the C-terminal side of UTI
  • a codon frequently used in E. coli is selected and is one of the translation termination signal codons at the 3 'end of the structural gene.
  • TGA is continuously arranged, a Trp terminator is added downstream of the TGA, and a restriction enzyme recognition site (a Kpnl cleavage site at the 5 ′ end) is provided at both ends for construction of the starting gene.
  • SEQ ID NO: 2 shows c was then designed and the genes having a structure, separated at the site showing the gene in FIG. 1, SEQ ID NO [corresponding sequences of Figure 1 (1) to (10)]: SEQ ID NO: 3 :
  • Each oligonucleotide (10 kinds in total) having the nucleotide sequence shown in Fig. 12 was chemically synthesized. The synthesis of these oligonucleotides was carried out by the phosphoramidite method using a fully automatic DNA synthesizer (Model 381A, Applied Biosystems).
  • a polyacrylamide gel electrophoresis (gel concentration: 20%) containing 7M urea was performed, and the gel was stained with ethidium / methylene chloride, and the target orange was generated on a long-wavelength (365 nm) ultraviolet ray generator. A band part corresponding to a gonucleotide was cut out and finely crushed.
  • Add 1 ml of DNA elution solution [20 mM Tris-HCl (pH 8.0), 1.5 mM EDTA] to this disrupted product, shake at 37 ° C, remove the supernatant obtained by centrifugation. Purification was performed using a salt column to obtain a synthetic oligonucleotide solution.
  • HCl pH 7.6
  • lOmM MgCl 2 the solution was treated at 90 ° C. for 5 minutes, and annealing was performed by slowly cooling to room temperature.
  • the synthesized DNA fragments were separated by urea-containing polyacrylamide gel electrophoresis (gel concentration: 10%) and purified by the synthetic oligonucleotide purification described above. Purification was performed in the same manner as in the above.
  • the plasmid PTV118N (Takara Shuzo) was digested with restriction enzymes EcoRI and Kpnl, and the linearized fragment was separated by agarose gel electrophoresis, and a gel containing the target DNA band was cut out.
  • the gel block was frozen at -80 ° C for 1 hour, and then rapidly heated to 37 ° C, and filtered through a 0.1 Hi pore centrifugal filter (Millipore). 'After extracting the filtrate with phenol, ethanol precipitation was performed, and the linearized plasmid was purified and recovered.
  • the amino acid sequence of the signal peptide of Escherichia coli outer membrane protein A was coded by annealing the single-stranded DNAs shown in SEQ ID NO: 13 and SEQ ID NO: 14. Then, a double-stranded DNA fragment having an _li ⁇ HI sticky end on the 5 'end of the coding strand and a blunt end on the 3' end was constructed using the method described above.
  • a 0.25 kb DNA fragment containing the synthetic gene generated by digestion with Rsal and J ⁇ RI was ligated between the restriction enzyme _ ⁇ site of PTV118N and the EcoRI site in the manner described above.
  • the reaction solution was further transduced into E. coli (JM109) according to the method described above, and some of the colonies grown on the LB-agar plate containing ampicillin were cultured. Mid DNA was extracted.
  • PCD17R15 The plasmid that was confirmed to have the desired gene by analysis of the agarose electrophoresis pattern after restriction enzyme digestion and nucleotide sequence analysis by the dideoxy method was named PCD17R15.
  • SEQ ID NO: ⁇ Mi consists 18 residues shown in 15 Roh It was selected 561- 1 2 ⁇ 1] 1 4 1 realm containing acid sequence.
  • the peptide having the amino acid sequence was synthesized by a solid-phase method using a fully automatic peptide synthesizer (Model 431A, Applied Biosystems). Cleavage of the synthesized peptide from the support was performed according to the manual of Applied Biosystems, and purification of the cleaved synthetic peptide was performed by reversed-phase chromatography.
  • Escherichia coli (JM109) holding PCD17IU5 was added to 100 ml / ml of ampicillin in 20 ml of TB medium (1.2% pactriptone (Dco), 2.4% yeast extract 10), 2.31 g / l of KH 2 P0 4, 12.54g / l of K 2 HP0 4, 0.4% of glycerol was inoculated into (pH 7.0)], 37. C, pre-cultured.
  • This pre-cultured solution was added to 400 ml of TB medium for main culture so that it became 0D G00nm vigorously to 0.1, and the cells were cultured at 37 ° C with shaking.c When the 0D G00nm reached 0.5, the final concentration was Isopropyl thiopiogalactoside (IPTG; Nacalai Tesque, Inc.) was added to the culture solution to 100 M, and the culture was continued until late in the logarithmic growth phase.
  • IPTG Isopropyl thiopiogalactoside
  • the cells were collected by centrifugation, and the cells were washed with a buffer consisting of 50 mM Tris-HCl (H 8.0), 1 mM EDTA, and 50 mM NaCl.
  • the washed cells were resuspended in 1/20 volume of the same buffer of the original culture, and the cells were disrupted using an ultrasonic generator under ice-cooling.
  • the disrupted cells were centrifuged to obtain recombinant HI-8 in the form of a precipitate.
  • the precipitate was dissolved in an aqueous solution of 20 mM Tris-HCl (pH 8.5) containing 8 M urea to obtain a crude purified recombinant HI-8 solution.
  • TBS buffer Tris-HCl ( ⁇ 7.6) buffer containing 150 mM NaCl
  • the cells were incubated at 4 ° C for 2 hours in a TBS buffer containing alkaline phosphatase-labeled goat anti-heron IgG solution (Biorad) and 1% skim milk.
  • TBS buffer containing 0.05% Tween 20
  • Tris-HCl pH 9.5 again containing washed thoroughly PVDF membrane, which developing solution alkaline phosphatase ⁇ synthetase [5 ml of LOMM NaCl, the 0.5 mM MgCl 2 )
  • the solution was prepared by dissolving 20 1 of nitrotetrazolium blue solution (a solution of nitrotetrazolium blue (Dohin Chemical Co., Ltd.) at a concentration of 50 mg / ml in 70% dimethylformamide solution) and 20 ⁇ 1.
  • the recombinant HI-8 expressed by PCD17R15 was named R15.
  • Example 3 SDS-polyacrylamide gel electrophoresis was performed in the same manner as in Example 3, and Furthermore, electroblotting was performed to transfer the expression peptide onto a PVDF membrane.
  • the PVDF membrane section retaining this expression peptide was applied to a protein sequencer (Model 477A, Applied Biosystems), and N-terminal amino acid sequence analysis was performed.
  • a protein sequencer Model 477A, Applied Biosystems
  • N-terminal amino acid sequence analysis was performed.
  • about 80% of this sample showed the N-terminal amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1, as designed, and the cleavage of signal peptide was also intended.
  • Mutant K registered trademark
  • Kit Tekara Shuzo
  • Escherichia coli (CJ236) carrying PCD17R15 was infected with helper phage M13K07 to obtain a -chain type PCD17R15.
  • the synthetic oligonucleotide having the sequence of SEQ ID NO: 16 was phosphorylated at the 5 'end of the oligonucleotide, annealing was performed, and the DNA polymerase and DNA polymerase in the kit were annealed.
  • the complementary strand was synthesized with Ise.
  • This synthetic DNA was introduced into Escherichia coli (BMH71-18mutS), cultured at 37 ° C for 1 hour, infected with M13K07, and further cultured at the same temperature for 16 to 20 hours. After the culture, the culture supernatant obtained by centrifuging the culture is diluted appropriately and added to the culture of Escherichia coli (MV1184) .After 10 minutes, an appropriate amount is plated and incubated at 37 ° C. A colony was formed. Select some of the grown colonies and The nucleotide sequence of the plasmid mutation introduction site of these bacteria was confirmed by the didoxy method. The plasmid into which the desired mutation was introduced was named PCD17IU5G3.
  • Synthetic oligonucleotides were applied to single-stranded PCD17R15G3 to perform site-specific mutagenesis to produce modified amino acid residues.
  • the synthetic oligonucleotide to be mutated has a sequence shown in SEQ ID NO: 17 to SEQ ID NO: 22.
  • the names of the primers (synthetic oligonucleotides) corresponding to the groups and their base sequences are specifically described.
  • amino acid modifications at the 15th position are described at the top of the modified amino acids. For example, those in which the third and fifteenth positions from the N-terminus have been modified to glycine and isoleucine, respectively, are referred to as I15G3, and those in which the eleventh position has been modified to glutamic acid are referred to as I15G3E11.
  • the modification was performed in the same manner as in Example 4 using a Mutan K kit, and the necessary amino acid residues were modified. In the modification of two or more amino acid residues, further residue conversion was repeated based on the mutated plasmid in which the modification of one residue was completed. In addition, the nucleotide sequence of each mutation-introduced site in the obtained mutant plasmid was confirmed by the dideoxy method. The thus obtained mutant plasmid was collectively referred to as PCD17XN based on the type (X) of the modified amino acid residue and the modification site (N: the number of residues from the N-terminal).
  • the present inventors using the above-described modification method, pCD17I15G3, pCD17L15G3, pCD17V15G3, pCD17V15G3El U pCD17V15G3Q46, pCD17V15G3EllQ46, pCD17I 15G3B11, pCDITI 15G3Q46,
  • E. coli cells transfected with any of the above plasmids, pCD17I15G3, pCD17V15G3Q46, PCD17V15G3E11Q46, pCD1715G3Q46, and pCD17115G3E11Q46 are prepared. Deposited on February 17, 1993 with the Institute of Biotechnology, Institute of Industrial Technology, located at 1-3 1-3 Babahigashi. The details are as follows.
  • Escherichia coli JM109-PCD17I15G3 (FERM BP-4556), which carries plasmid PCD17115G3;
  • Escherichia coli JM109-PCD17V15G3Q46 (FERM BP-4557), which carries plasmid PCD17V15G3Q46;
  • Escherichia coli JM109-PCD17V15G3E11Q46 (FERM BP-4560) which carries plasmid PCD17V15G3E11Q46;
  • Escherichia coli JM109-PCD17I15G3Q46 (FERM BP-4558), which carries plasmid PCD17I15G3Q46;
  • this crude purified solution was dialyzed against a 20 mM ammonium acetate (pH 5.5) solution, centrifuged, and the supernatant was subjected to ion exchange chromatography using a Mono-Q column (Pharmacia). Graphite [Acetate containing 0.5M NaCl from a 20 mM ammonium acetate (pH 5.5) solution Elution with a linear concentration gradient for 10 minutes into an ammonia (pH 5.5) solution].
  • Example 8 Measurement of inhibitory activity on human neutrophil elastase and esophageal trypsin
  • the measurement of the Ki value for human neutrophil elastase was performed as follows.
  • the mixture was excited at 365 nm for 0 to 3 minutes, and the 450nrn fluorescence was measured by a spectrofluorometer (F-3000, manufactured by Hitachi, Ltd.). ), And the initial reaction rate was determined. For each substrate concentration, a graph was created with the reciprocal of the initial reaction rate on the vertical axis and the sample concentration on the horizontal axis, and the Ki value was determined from the intersection of the obtained three straight lines.
  • HCK Nacalai Tesque HCK Nacalai Tesque solution ( (Containing 10% DMS0) were added at 350 ° C and reacted at 30 ° C. The change in absorbance with time at 405 nm was recorded, and the initial reaction rate of the enzyme was determined from this. Then, the Ki value for tribcine was determined in the same manner as in the case of human neutrophil elastase described above.
  • Ki values of (R15), recombinant H8 (R15G3), and modified ⁇ -8 of the present invention for human neutrophil elastase are shown in Table 2 below.
  • the modification of the combination of glutamic acid at position 11 and glutamine at position 46 adds K i to human neutrophil elastase. And Turkey can be reduced value until the 6 ⁇ 8 x 10_ 1 I was found.
  • the novel elastase inhibitory peptide obtained in the present invention has potent human neutrophil elastase inhibitory activity, while it has natural elastase inhibitor and chemical synthesis.
  • Pulmonary emphysema, etc. because they are expected to have higher resistance to oxidation, lower immunogenicity and lower toxicity to humans compared to low molecular weight Erasase inhibitors It is considered to be extremely useful as a therapeutic or prophylactic agent for elastase-induced diseases.
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • CTAGAGCCCA GCCCGCCTAA TGAGCGGGCT TTTTTTTGAA CAAAAGGCGG 50
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • SEQ ID NO forms a complementary strand with the sequence shown in 13.
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • amino acid at position 18 of recombinant HI-8 in which the amino acid at position 15 has been changed to isoleucine is further converted to phenylalanine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

On produit un nouveau peptide présentant une activité inhibitrice de l'élastase utilisé pour le traitement ou la prophylaxie de maladies provoquées par l'élastase en modifiant partiellement l'acide aminé, constitutif de HI-8 présent sur le côté de l'extrémité C d'un inhibiteur de la trypsine urinaire humaine et présentant une activité inhibitrice de la trypsine. L'invention concerne également un gène codant pour le peptide, un plasmide contenant ledit gène, un microbe recombiné contenant ledit plasmide et un procédé de production dudit peptide au moyen dudit microbe recombiné.
PCT/JP1994/000284 1993-02-22 1994-02-21 Nouveau peptide presentant une activite inhibitrice de l'elastase et son procede de production WO1994019371A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU61151/94A AU676019B2 (en) 1993-02-22 1994-02-21 Novel peptide having esterase inhibitory activity and methodof producing same
US08/325,243 US5541288A (en) 1993-02-22 1994-02-21 Peptide having elastase inhibitory activity and producing method thereof
KR1019940703771A KR100213662B1 (ko) 1993-02-22 1994-02-21 엘라스타제 저해활성을 갖는 신규 펩티드 및 그의 제조방법
EP94907668A EP0643075A4 (fr) 1993-02-22 1994-02-21 Nouveau peptide presentant une activite inhibitrice de l'elastase et son procede de production.
CA002134240A CA2134240C (fr) 1993-02-22 1994-02-21 Nouveau peptide agissant comme inhibiteur de l'elastase et mode de production d'obtention de cette molecule

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP5031758A JP2769083B2 (ja) 1993-02-22 1993-02-22 エラスターゼ阻害活性を有する新規ペプチドおよびその製造方法
JP5/31758 1993-02-22

Publications (1)

Publication Number Publication Date
WO1994019371A1 true WO1994019371A1 (fr) 1994-09-01

Family

ID=12339931

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1994/000284 WO1994019371A1 (fr) 1993-02-22 1994-02-21 Nouveau peptide presentant une activite inhibitrice de l'elastase et son procede de production

Country Status (7)

Country Link
US (1) US5541288A (fr)
EP (1) EP0643075A4 (fr)
JP (1) JP2769083B2 (fr)
KR (1) KR100213662B1 (fr)
AU (1) AU676019B2 (fr)
CA (1) CA2134240C (fr)
WO (1) WO1994019371A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1734121A3 (fr) * 1994-12-16 2008-09-10 Dyax Corporation Domaines de Kunitz dérives de l'homme obtenus par génie génétique et inhibant l'élastase des neutrophiles chez l'homme

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060134087A1 (en) * 1988-09-02 2006-06-22 Dyax Corp. ITI-D1 Kunitz domain mutants as hNE inhibitors
US7078383B2 (en) * 1988-09-02 2006-07-18 Dyax Corp. ITI-D1 Kunitz domain mutants as HNE inhibitors
US5650394A (en) * 1993-11-04 1997-07-22 Adeza Biomedical Use of urinastatin-like compounds to prevent premature delivery
NZ331540A (en) 1996-03-11 1999-10-28 Bayer Ag Human bikunin
US20070140979A1 (en) * 1998-12-22 2007-06-21 Bayer Aktiengesellschaft Method for accelerating the rate of mucociliary clearance
EP1374891B1 (fr) * 1998-12-22 2008-09-10 Bayer Aktiengesellschaft Technique permettant d'accélérer le débit de clairance mucociliaire
US6989369B2 (en) 2003-02-07 2006-01-24 Dyax Corp. Kunitz domain peptides
JP6574431B2 (ja) 2014-02-24 2019-09-11 タケダ ゲー・エム・ベー・ハーＴａｋｅｄａ ＧｍｂＨ Ｕｔｉ融合タンパク質

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK0401508T3 (da) * 1989-05-13 1995-05-15 Bayer Ag Proteaseinhibitorer, fremgangsmåde til deres fremstilling samt lægemidler med indhold deraf
AU641568B2 (en) * 1990-11-13 1993-09-23 Mochida Pharmaceutical Co., Ltd. Polypeptide,DNA fragment encoding the same and process for producing the same, and enzyme inhibition process, drug composition and methods of treating using the same
ATE243710T1 (de) * 1991-03-01 2003-07-15 Dyax Corp Hemmstoffe für menschliche neutrophile elastase und menschliches kathepsin g
US5451659A (en) * 1991-11-08 1995-09-19 Mochida Pharmaceutical Co., Ltd. Polypeptide, DNA fragment encoding the same, drug composition containing the same and process for producing the same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 105, No. 3, the Abstract No. 17700n; H. OHNISHI et al.: "Pharmacological Activities of a Trypsin Inhibitor, Urinastatin (Mivaclid)"; & Oyo Yakuri, 1986, 31(3), p. 663-75. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1734121A3 (fr) * 1994-12-16 2008-09-10 Dyax Corporation Domaines de Kunitz dérives de l'homme obtenus par génie génétique et inhibant l'élastase des neutrophiles chez l'homme

Also Published As

Publication number Publication date
JPH06247998A (ja) 1994-09-06
AU6115194A (en) 1994-09-14
KR950701345A (ko) 1995-03-23
AU676019B2 (en) 1997-02-27
US5541288A (en) 1996-07-30
EP0643075A4 (fr) 1997-02-26
CA2134240C (fr) 2000-08-01
KR100213662B1 (ko) 1999-08-02
EP0643075A1 (fr) 1995-03-15
JP2769083B2 (ja) 1998-06-25
CA2134240A1 (fr) 1994-08-23

Similar Documents

Publication Publication Date Title
JP2894354B2 (ja) 組換えｄｎａ技術によつて産生されたウシ膵臓トリプシン阻害剤の変異型、それらの方法、発現ベクターおよび組換え宿主、およびそれらの製薬学的使用
Koebnik et al. Insertion derivatives containing segments of up to 16 amino acids identify surface-and periplasm-exposed regions of the FhuA outer membrane receptor of Escherichia coli K-12
JPH07143883A (ja) リパーゼ遺伝子及び変異体リパーゼ
FI97139C (fi) Menetelmä haiman erittämän trypsiini-inhibiittorivarianttien valmistamiseksi ja menetelmässä käytettävä DNA, vektori ja isäntäsolu
CA2018592C (fr) Polypeptides et polypeptides analogues avec une activite inhibitrice contre l'elastase humaine
KR20000019788A (ko) 대장균 엔테로톡신ⅱ 신호펩티드의 변형체와 인체성장호르몬의융합단백질을 발현하는 재조합 미생물 및 그를 이용한 인체성장호르몬의 제조방법
JPH07506334A (ja) ヒトＫｕｎｉｔｚ型プロテアーゼ阻害剤変異体
JPH08504568A (ja) ウシ膵トリプシン阻害因子から誘導される因子ＶＩＩａ−組織因子複合体の阻害因子
JPS62500074A (ja) バチルス　スリンギエンシス　結晶タンパク質遺伝子毒素セグメント
WO1994019371A1 (fr) Nouveau peptide presentant une activite inhibitrice de l'elastase et son procede de production
US5496713A (en) Process for producing 20 kD human growth hormone
JPH04166085A (ja) 新規プロテアーゼ
WO1986004351A1 (fr) Prourokinase humaine stabilisee
US5589360A (en) Polypeptide, DNA fragment encoding the same, drug composition containing the same and process for producing the same
WO1989001513A1 (fr) Prourokinase a action rapide
JPH04228078A (ja) 微生物由来ペルオキシダーゼ遺伝子
JPWO2004113535A1 (ja) 凝集抑制作用を有するシヌクレイン変異体
JP2878341B2 (ja) 人工機能性ポリペプチド
US5993807A (en) Truncated aspartase enzyme derivatives and uses thereof
CA2166298A1 (fr) Famille de peptides appeles xenoxines
JP2674796B2 (ja) エラスターゼをコードする遺伝子
JP2782372B2 (ja) サイクロデキストリングルカノトランスフェラーゼ遺伝子、サイクロデキストリングルカノトランスフェラーゼの製造法及びプラスミド
US20030049686A1 (en) Method for manufacturing mutnat library of proteins with various sizes and sequences
JPWO2005007686A1 (ja) Ｒｎａ結合ペプチド
JPH04258294A (ja) 分泌ベクター、該ベクターで形質転換した微生物及び該微生物から産生される産物の製造法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA KR US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

WWE Wipo information: entry into national phase

Ref document number: 2134240

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1994907668

Country of ref document: EP

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 08325243

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1994907668

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1994907668

Country of ref document: EP